This vector is under T7 promoter so you will need IPTG to trigger the expression. Have you added the IPTG at a proper timing? How do your cells grow? Do they grow dense? How do you break the cells? Sonication? So your protein should have 6Xhis tag, do you use nickle beads to purify it?Have you try to lyse some cells, run a protein gel and perform western blot with anti-His antibody? In this way you can see in small amount whether the protein is expressed before trying to collect large amount.

It would be nice if you provide more details of the system, and method. It seems that you may load various amounts and run WB.

Heng Liang

Thank you for your response. I did all the things you mentioned, except Western Blot, but unfortunately, protein expression was not observed.

In this work:

The final concentration of IPTG is used from 0.5 to 1 mM

The cells grow at about 180 RPM and IPTG is added at a concentration of 0.6 to 0.8

I break the cells with sonication and detected the protein bond using polyacrylamide gel electrophoresis (SDS PAGE).

What strain of e coli r u using? Since cloning a eukaryotic gene then you might be facing codon usage problems

What temperature are you growing your cells? You may try growing at lower temperature. Sometimes that will help.

Have you checked that your cloned gene is in the correct reading frame? Other possibilities:

the protein is toxic to your bacteria cells

the protein forms insoluble clumps or aggregates

your IPTG isn't inducing expression - expired/wrong dose/added at wrong time

Many factors effect on cloning :

1- Vectors are accept by host or not (must be experimented) and size of gene

2- The gene integrated with host DNA or not

3- Some specific condition must be applied

If you are sure on cloning, transformation,...It may be for the wrong dose of IPTG or situation of storage of IPTG was wrong. Please used the fresh one.

Katie A Burnette

Thank you for your response.

Yes, according to the sequencing result, the gene is inserted correctly.

If the gene has a toxic effect on my bacteria, how can I eliminate this effect?

Afsaneh Sadre Momtaz

In the case of IPTG, I tested two different sources, but unfortunately, the results were similar.

If the gene is toxic, there are strategies you can try:

Article Expression of Highly Toxic Genes in E. coli: Special Strateg...

did you compare a growing curve? (with untransformed cells) sometimes the protein is toxic, and even though the promoter is not induced, its not uncommon a leak (more if you are using BL21 DE3 with T7), considering it is a relatively big protein. What method did you use to analyze expression? MAYBE, give a try extracting and concentrating with the His Tag and try a WB... to know if the construct is working at least

I have expressed 300+ proteins, both prokaryote and eukaryote, in BL21. Lack of expression can be due to just a few reasons. You have many sensible responese already. To add to that, my systematic troubleshooting process is as below:

1. Verify the DNA sequence of the expression vector. Is your insert in-frame with the his tag and the start codon? Is the promoter the right sequence? Is the ribosome binding site spaced optimally from the start codon (~7-8 bp)? Are there any frame shifts in the coding sequence of your insert? Are there rare codons for E. coli B in the coding sequence? Always codon-optimise. In 99% of cases codon optimisation and correcting errors in DNA sequence resolved the expression problems. If you can't reclone a codon optimised version of your gene then express it in Rosetta or similar strain with tRNAs for rare codons.

2. If the DNA sequence is fine, then test that the protein being expressed is not toxic. Transform a BL21AI strain with your plasmid and the expression vector with no insert as a control. BL21AI has the T7 polymerase under control of the arabinose inducible tight promoter. Compare the growth rates (A600) of control Vs your plasmid when not induced and when induced with several concentrations of arabinose. If the protein is toxic, two things can happen: (i) a large reduction in growth rate when the protein is induced or (ii) DNA rearrangements in the coding sequence or mutations in the promoter/RBS that eliminate or reduce expression - these can be determined by streaking for single colonies after induction is complete and sequencing the plasmid DNA from several colonies to check for mutations. Note that toxicity is apparent only if the protein is expressed; lack of symptoms of toxicity can also be due to zero expression in the first place. A less dramatic difference in growth rate between expressing and non-expressing cultures should be apparent even for non-toxic protein.

3. If protein is toxic to E.coli, then there are alternative strategies. (i) In the AI strain, grow to a high OD and blast with arabinose for a short time (1h) then harvest etc. (ii) add a signal sequence so that the protein is rapidly secreted into the medium (iii) Change to yeast, pichia or other expression system (iv) in vitro expression (expensive and not scalable) (v) express smaller domains of your protein (not always feasible).

Note also that 85 kDa is a large protein. In the pET/BL21 expression system, protein yield is inversely proportional to size i.e. the larger a protein the lower the yield. You may need a more sensitive detection method such as Western Blotting to see your expressed protein.

Deepan Shah Thank you for your response.

The answer was very helpful

Only I did not understand some of your answers well can you explain more about them?

1) Ribosome binding site spaced optimally from the start codon (~7-8 bp)

( I am not able to manipulate this site during gene cloning )

2) BL21AI has the T7 polymerase under control of the arabinose inducible

(I using strain bl21 DE3.

Do you mean the arabinose inducer is IPTG?

Can the method used for A1 be used for DE3?)

3) Grow to a high OD

(What OD do you suggest?)

4) Blast with arabinose for a short time (1h) then harvest etc

(Can you explain this part more?)

Thank you

Hello,

Some clarification:

1) Ribosome binding site spaced optimally from the start codon (~7-8 bp) ( I am not able to manipulate this site during gene cloning )

This is only an issue if your start codon is somewhere distal to the cloning site. 2) BL21AI has the T7 polymerase under control of the arabinose inducible (I using strain bl21 DE3. Do you mean the arabinose inducer is IPTG? The AI strain is different from BL21DE3 in that it has an arabinose-indicible T7 RNA Pol. It is induced by arabinose instead of IPTG. The advantage of this is that the ara promoter is not as leaky as the tac promoter. 3) Grow to a high OD (What OD do you suggest?)

You have to optimise this. It will depend on the growth conditions (medium) and the protein of interest. 4) Blast with arabinose for a short time (1h) then harvest etc (Can you explain this part more?) By "blast" I mean induce with arabinose for a short time. This is a good method for toxic genes.